Capsaicinoids are products of the phenylpropanoid pathway, a central and ubiquitous in plant secondary metabolism. The products of this pathway are diverse, perform a multitude of functions in the plant, and provide a multitude of pharmaceutical agents. There are heritable differences in the amount and type of capsaicinoids that accumulate, these differences are controlled by other genes, whose nature is also unknown. Our long-term goal is to understand the genetic, biochemical, physiological and environmental influences on the biosynthesis of capsaicinoids. The first specific aim of this proposal is to confirm the biochemical functions proposed for 5 cDNAs isolated from a library of habanero placental transcripts. The genes encoded by these cDNAs are predicted to be on the capsaicinoid pathway based on sequence similarities detected using blastp alignments. These functions will be confirmed using in vitro assays on recombinant proteins will be used to monitor accumulation of the protein in chile and in transgenic plants. The second specific aim of this proposal is to isolate genomic clones of those cDNA sequences demonstrated to encode capsaicinoid biosynthetic activities. Potentially 9 genes will be characterized in this manner from two chile genotypes differing in degree of pungency. The promoter elements of the coordinately regulated genes will be compared to identify cis-elements for transcriptional control. The coding region of pairs of alleles will be compared to determine if significant differences are associated with different pungency levels. To meet these objectives, an excellent collection of genetic, biochemical, and molecular, biological tools has been assembled. Well characterized chile germplasm, including lines differing in pungency and non-pungent mutants in several different backgrounds is available. The patterns of gene expression for several genes predicted to encode different biosynthetic enzymes has been described. Transcript abundances for genes on the capsaicinoid pathways are proportional to the concentration of capsaicinoids accumulated in the placenta. Analytical procedures to monitor the concentration of specific intermediates on the capsaicinoid pathway have been developed. Hypotheses regarding the control of expression of capsaicinoid biosynthesis will be tested: (1) The promoters of coordinately-regulated capsaicinoid genes have common cis-elements. (2) Degrees of pungency in chile is the result of differential accumulation of biosynthetic enzymes, not due to different catalytic activities.